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. 2025 Jul 23;10(35):39923–39932. doi: 10.1021/acsomega.5c04299

Dissolvable Microneedles Embedded with Chitosan Nanoparticles for Transdermal Delivery of Pramipexole Dihydrochloride in Parkinson’s Disease

Kankanit Phetporkha †, Chonticha Saisawang ‡, Nattaphong Rattanavirotkul §, Soracha D Thamphiwatana †,*, Doungdaw Chantasart ∥,*
PMCID: PMC12423965  PMID: 40949249

Abstract

Parkinson’s disease (PD), a progressive neurodegenerative disorder, presents significant challenges in long-term management due to the limitations of conventional oral dopaminergic therapy. Pramipexole dihydrochloride (Px), though clinically effective, is associated with gastrointestinal side effects and reduced compliance, especially in patients with dysphagia. To address these challenges, we developed a transdermal drug delivery system that integrates Px-loaded chitosan nanoparticles within dissolvable microneedle arrays. This hybrid platform combines the sustained release capability of polymeric nanoparticles with the minimally invasive administration of microneedles. The system is designed to penetrate the stratum corneum, dissolve upon insertion, and release Px nanoparticles for sustained dermal absorption. The microneedle arrays exhibit structural uniformity, mechanical robustness, and efficient skin insertion. The nanoparticles, in turn, enable controlled drug release while maintaining biocompatibility. Biological assessments confirmed the safety of the formulation, showing compatibility with human dermal cells and red blood cells. Moreover, ex vivo permeation studies demonstrated enhanced delivery of Px through human skin compared to free drug formulations. This work offers a promising solution for sustained, noninvasive delivery of dopaminergic therapy, with potential to improve clinical outcomes and quality of life in PD.

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1. Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons and the accumulation of Lewy bodies. Clinically, PD manifests as tremor, rigidity, bradykinesia, and postural instability. As the disease advances, patients often experience nonmotor symptoms such as cognitive impairment and neuropsychiatric complications, all of which significantly diminish quality of life. Current pharmacological interventions primarily aim to alleviate symptoms through dopaminergic agents, including dopamine precursors and dopamine agonists.

Pramipexole, a nonergot dopamine agonist with high affinity for D3-type dopamine receptors, is widely used for its neuroprotective potential and favorable side effect profile relative to levodopa. It has demonstrated efficacy in activating dopaminergic pathways and slowing the progression of PD. − The dihydrochloride salt form of pramipexole (Px) is highly water-soluble and possesses a relatively low molecular weight (284.2 g/mol), making it a suitable candidate for alternative delivery routes. However, the therapeutic efficacy of Px is limited by its conventional oral formulation, which is often associated with gastrointestinal side effects, and difficulty in administration for patients experiencing dysphagia. These drawbacks can compromise treatment adherence and clinical outcomes. −

To address these limitations, transdermal drug delivery systems have emerged as a promising alternative to bypass the gastrointestinal tract and deliver drugs directly into systemic circulation. Microneedles (MNs), in particular, offer a minimally invasive platform capable of breaching the stratum corneum to facilitate drug delivery. This approach improves bioavailability, allows sustained and controlled drug release, and enhances patient compliance, especially in chronic conditions such as PD. −

Furthermore, encapsulating Px in polymeric nanoparticles may improve stability, prolong systemic circulation, and enable targeted delivery to dopaminergic neurons. − When combined with microneedle technology, this hybrid system may overcome the limitations of oral delivery while enabling precise and sustained transdermal administration of Px. −

In this study, we present the development of a novel transdermal delivery platform combining pramipexole-loaded chitosan nanoparticles with dissolvable microneedle arrays (Figure ). We aimed to enhance transdermal permeation, mechanical properties, and biocompatibility of the microneedle system. This approach offers the potential to address critical limitations of oral PD therapy, reduce systemic side effects, and improve patient adherence, thereby contributing to more effective disease management.

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Schematic illustration of Px NPs MNs for transdermal delivery. Px NPs are embedded within dissolvable microneedles, which penetrate the skin, dissolve, and release Px NPs into deeper skin layers for systemic absorption.

2. Materials and Methods

2.1. Materials, Skin and Whole Blood

Low molecular weight chitosan, sodium tripolyphosphate (TPP), carboxymethylcellulose sodium salt (CMC, low viscosity), poly­(vinyl alcohol) (PVA; molecular weight (M W) 30,000–70,000 Da), and triethylamine (TEA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). High-performance liquid chromatography (HPLC)-grade acetonitrile was obtained from Supelco (Bellefonte, PA, USA), and glacial acetic acid was supplied by QRëC (Auckland, New Zealand). Px was synthesized in-house as previously reported. , Polydimethylsiloxane (PDMS) microneedle molds used for fabrication were procured from Blueacre Technology Ltd. (Dundalk, Ireland). All other chemicals and reagents were of analytical grade.

Excess human abdominal skin was obtained postoperatively from the Plastic Surgery Center, Department of Surgery, Yanhee International Hospital (Bangkok, Thailand), and used as a model membrane for microneedle studies. The skin was rinsed with phosphate-buffered saline (PBS, pH 7.4), cleaned of residual subcutaneous tissue, blotted dry with lint-free tissue, and stored at −20 °C in aluminum foil until use.

Fresh human blood was collected from a healthy adult donor into sodium citrate tubes for use in hemocompatibility assays. The use of both human skin and blood samples was approved by the Committee on Human Rights Related to Human Experimentation, Mahidol University, Thailand (COA No. MU-MOU 2024/125.2603).

2.2. Nanoparticle Fabrication and Characterization

2.2.1. Fabrication of Px-Loaded Chitosan Nanoparticles (Px NPs)

Px NPs were prepared using the ionic gelation method with TPP as the cross-linking agent. Chitosan was dissolved in 1% (v/v) acetic acid to a final concentration of 1 mg/mL and stirred overnight at room temperature. The solution was then filtered to remove any undissolved particulates, and the pH was adjusted to 5.0 using 1 M sodium hydroxide (NaOH). Separately, TPP was dissolved in deionized water at a concentration of 1 mg/mL, and the pH was adjusted to 2.0 using 1 M hydrochloric acid (HCl).

Px was mixed with the TPP solution at a drug input concentration ranging from 5% to 30% (w/w, relative to chitosan) and incubated under constant magnetic stirring for 15 min. This Px-containing TPP solution was then added dropwise into the chitosan solution at a volumetric ratio of 1:3 (TPP/chitosan) under continuous stirring. After an additional 5 min of mixing to facilitate cross-linking and nanoparticle formation, the nanoparticle suspension was centrifuged using a centrifugal filter device (M W cutoff: 10 kDa) (Amicon Ultra-4 10K MWCO, MilliporeSigma, MA, USA) to separate unencapsulated drug from the nanoparticle fraction.

2.2.2. Particle Size and Zeta Potential Analysis of Px NPs

The hydrodynamic diameter and zeta potential of Px NPs were measured using dynamic light scattering (DLS) on a HORIBA SZ-100 V2 nanoparticle analyzer. Samples were analyzed in triplicate at 25 °C, and the mean values were reported. Prior to measurement, each sample was appropriately diluted with deionized water to avoid multiple scattering effects. Results are presented as the average particle size (z-average) and zeta potential ±standard deviation.

2.2.3. Determination of Encapsulation Efficiency (%EE) and Drug Loading (%DL) of Px NPs

The %EE and %DL of Px NPs were determined using an indirect quantification method. Unencapsulated drug was separated from the nanoparticle suspension using a centrifugal filtration device at 4500g for 30 min. This process yielded a concentrated nanoparticle fraction (retentate) and a filtrate containing free Px (supernatant). To quantify the unencapsulated drug, the supernatant was freeze-dried to remove residual acetic acid and then reconstituted in 1 mL of methanol. The resulting solution was diluted 1:10 with the HPLC mobile phase, consisting of 0.5% TEA and acetonitrile (97:3, v/v). Samples were filtered through a 0.2 μm nylon syringe filter prior to analysis by HPLC.

2.2.4. In Vitro Cumulative Drug Release Study

The in vitro drug release profile of Px NPs was evaluated using a dialysis method in PBS, pH 7.4 at 37 °C to simulate physiological conditions. A 2 mL aliquot of concentrated Px NP suspension was transferred into a presoaked dialysis membrane (M W cutoff: 12–14 kDa) and immersed in 200 mL of PBS, pH 7.4 in a beaker maintained at constant temperature (37 °C) under gentle stirring. At predetermined time intervals, 1 mL samples of the release medium were withdrawn and immediately replaced with an equal volume of fresh prewarmed PBS, pH 7.4 to maintain sink conditions and constant volume. The collected samples were filtered using a 0.2 μm nylon syringe filter and analyzed for pramipexole content using HPLC as previously described.

2.2.5. Morphological Characterization of Px NPs

The morphology of Px NPs was examined using field emission scanning electron microscopy (FE-SEM; JSM-7610FPLUS, JEOL Ltd., Tokyo, Japan). Freeze-dried Px NP samples were mounted on carbon adhesive tape affixed to aluminum stubs and sputter-coated with a thin layer of gold using an autofine coater (10 mA, 60 s) to enhance surface conductivity. The coated specimens were then imaged under FE-SEM to observe particle shape, surface topology, and nanosphere morphology.

2.3. Cytotoxicity and Neuroprotective Effects of Px NPs on SH-SY5Y Cells

The human neuroblastoma cell line SH-SY5Y was cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12), supplemented with 15% fetal bovine serum (FBS), 50 U/mL penicillin, 50 μg/mL streptomycin, and 1% nonessential amino acids. Cells were maintained at 37 °C in a humidified atmosphere of 5% carbon dioxide (CO2) until reaching the appropriate confluence for experimentation. For all assays, cells were seeded in 96-well plates at a density of 4 × 104 cells per well and incubated overnight. To evaluate potential cytotoxicity, SH-SY5Y cells were treated with Free Px or Px NPs at various concentrations for 1 h at 37 °C. After treatment, cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium- bromide (MTT) assay. The MTT reagent was added, and following incubation, the resulting formazan crystals were solubilized and quantified by measuring absorbance at 540 nm. Cell viability was expressed as a percentage relative to untreated control cells. To assess neuroprotective effects, SH-SY5Y cells were pretreated with Free Px or Px NPs at the same concentrations for 1 h under same incubation conditions. After removing the treatment media, cells were exposed to 1 mM hydrogen peroxide (H2O2) for 15 min to induce oxidative stress. Cell viability was then measured using the MTT assay as described above. The protective effect was determined by comparing treated groups to H2O2-injured controls.

2.4. Microneedle Fabrication and Characterization

2.4.1. Fabrication of Px-Loaded Nanoparticle-Embedded Microneedles

Dissolvable microneedle arrays were fabricated using CMC and PVA as the matrix-forming polymers. Blank microneedles (B MNs), microneedles containing free Px (Free Px MNs) and Px-loaded chitosan nanoparticles microneedles (Px NPs MNs) were prepared using the same protocol. Prior to fabrication, Px NPs were concentrated using a centrifugal filter unit. The concentrated nanoparticles were then mixed with an aqueous solution containing 15% w/w CMC and 20% w/w PVA in a 1:1 weight ratio. The resulting microneedle formulation was poured into PDMS molds and centrifuged at 4000g for 15 min to eliminate air bubbles and ensure complete cavity filling. The filled molds were dried under desiccation with silica gel at room temperature for 24 h to ensure complete solidification. Following drying, microneedle patches were carefully demolded and the final weight of each patch was recorded. Free Px MNs were prepared using the same procedure, substituting pramipexole for the nanoparticle suspension. B MNs were fabricated identically, without the addition of Px or Px NPs.

2.4.2. Skin Insertion Efficiency (% Insertion)

The insertion efficiency of microneedles was evaluated using full-thickness human skin (FTS) as a model membrane. Three types of microneedle patches were tested: B MNs, Px NPs MNs, and Free Px MNs. FTS was mounted and stretched over a PDMS support, and a 1 × 1 cm2 microneedle patch was applied using a custom acrylic applicator with uniform force for 30 s, followed by patch removal. To visualize and quantify microneedle penetration, 400 μL of 1% (w/v) methylene blue solution was applied to the skin surface and allowed to stain for 30 min. Excess dye was removed by rinsing the skin three times with 2 mL of PBS, pH 7.4. Penetration sites were visualized as distinct blue spots, and the percentage of successful insertions was calculated using the following equation

%insertionefficiency=numberofbluespotobservednumberofneedles(121)×100

2.4.3. Mechanical Properties of Microneedles

The mechanical strength of B MNs, Px NPs MNs, and Free Px MNs was evaluated using a texture analyzer (TA.XTPlus, Stable Micro Systems Ltd., Surrey, UK). Each microneedle patch (1 × 1 cm2) was affixed to the cylindrical probe of the texture analyzer using double-sided adhesive tape. The probe was programmed to compress the patch against a flat stainless-steel plate at a constant speed of 0.5 mm/s until a maximum force of 32 N was reachedrepresenting the average manual force applied during microneedle administration, as reported in prior studies. The trigger force was set at 0.049 N, with both pretest and post-test speeds configured at 1 mm/s. Force–displacement data were recorded to assess microneedle deformation behavior under compressive load.

2.4.4. Skin Permeation Study

The transdermal permeation of pramipexole from microneedle formulations was evaluated using FTS mounted in Franz diffusion cells. Prior to experimentation, FTS samples were equilibrated in PBS, pH 7.4 at room temperature for 1 h. A 5 × 5 cm2 section of equilibrated skin was secured onto a PDMS support, and microneedle patches were applied following the same protocol as described in the insertion study. Following application, the skin and microneedle assembly were transferred to the donor chamber of a Franz diffusion cell with a diffusion area of 1.77 cm2. The stratum corneum faced the donor side and the dermal side was in contact with the receptor compartment. The receptor chamber was filled with 5 mL PBS, pH 7.4 and maintained at 32 °C using a thermostatically controlled water bath to mimic physiological skin temperature. The receptor medium was continuously stirred throughout the experiment to maintain sink conditions. An aliquot of 300 μL was withdrawn from the receptor compartment at predetermined time intervals (i.e., 8, 10, 12, 24, 36, 48, and 72 h), and an equal volume of fresh prewarmed PBS, pH 7.4 was immediately added to maintain volume consistency. Collected samples were filtered through 0.2 μm nylon syringe filters and analyzed for Px content via HPLC.

2.4.5. Microneedle Visualization

The morphology of microneedle arrays was examined using scanning electron microscopy (SEM; JSM-IT500LA, JEOL Ltd., Tokyo, Japan). Dried microneedle samples were mounted onto aluminum stubs using carbon adhesive tape and sputter-coated with a thin layer of gold using an autofine coater (10 mA, 60 s) to enhance surface conductivity. The coated samples were then imaged under SEM to assess microneedle geometry, surface integrity, and structural uniformity.

2.5. HPLC Analysis

Quantification of Px was performed using a reversed-phase HPLC method adapted from previously reported. The analysis was conducted on a Waters Alliance 2695 HPLC system (Waters Corporation, Milford, MA, USA), equipped with a Hypersil BDS C18 analytical column (150 mm × 4.6 mm, 5 μm particle size) and a corresponding guard column (Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase consisted of 0.5% (v/v) triethylamine in deionized water (pH adjusted to 6.0 using phosphoric acid) and acetonitrile in a 97:3 (v/v) ratio. Chromatographic separation was achieved under isocratic conditions at ambient temperature, with a flow rate of 1.0 mL/min and an injection volume of 50 μL. Detection of Px was carried out at 262 nm using a UV detector.

2.6. Biocompatibility Testing

2.6.1. Cytotoxicity Testing

Cytotoxicity of microneedle formulations was evaluated using human primary dermal fibroblasts (HDFn; ATCC, Manassas, VA, USA) in accordance with ISO 10993-5:2009 guidelines. HDFn cells were cultured in the recommended growth medium and maintained at 37 °C in a humidified 5% CO2 incubator until reaching confluence. To prepare extracts for testing, B MNs, Px NPs MNs, and B NPs MNs were incubated in complete culture medium at 37 °C for 24 ± 2 h. The resulting extracts were collected and diluted to various concentrations for use in cytotoxicity assays. HDFn cells were seeded into 96-well plates at a density of 1 × 104 cells per well and incubated overnight for cell attachment. The following day, cells were treated with the prepared microneedle extracts and incubated for 24 ± 2 h under standard culture conditions. Untreated wells containing complete culture medium served as the negative control, representing 100% cell viability, while wells treated with 0.1% Triton X-100 served as the positive control. Cell viability was assessed using the MTT assay. After incubation, MTT reagent was added to each well and the cells were incubated for an additional period to allow for formazan formation. The resulting formazan crystals were solubilized, and absorbance was measured at 540 nm using a microplate reader. All treatments were performed in triplicate, and cell viability was expressed as a percentage relative to the negative control using the following formula

%cellsviability=absorbanceofcellstreatedwithsamplesabsorbanceofuntratedcells×100

2.6.2. Hemolysis Activity Testing

The hemocompatibility of Px NPs and microneedle formulations was evaluated by assessing their hemolytic activity against human red blood cells (RBCs). Lyophilized formulations, B MNs, Px NPs MNs, and B NPs MNs, were preincubated in PBS, pH 7.4 at 37 °C for 24 ± 2 h prior to testing. Nanoparticle suspensions were prepared at concentrations of 0.25, 0.5, and 1 mg/mL. Fresh venous blood (10 mL) was collected from a healthy volunteer into citrate-coated tubes. The blood was centrifuged at 700g for 15 min to remove plasma, and the resulting packed RBCs were washed three times with PBS, pH 7.4 at the same speed. The washed RBCs were then diluted 10-fold in PBS, pH 7.4 to prepare the working suspension for hemolysis assays. For each test, 900 μL of the diluted RBC suspension was mixed with 100 μL of the sample solution. PBS, pH 7.4 served as the negative control (0% hemolysis), and 1% Triton X-100 solution was used as the positive control (100% hemolysis). Samples were incubated in a 37 °C water bath for 3 h, followed by centrifugation at 700g to pellet the cells. The supernatant from each sample was collected, and hemoglobin release was quantified by measuring the optical density (OD) at 540 nm using a microplate reader. The percentage of hemolysis was calculated using the following formula

%hemolysis=ODoftest−ODofnegativecontrolODofpositivecontrol−ODofnegativecontrol×100

2.7. Data Analysis

All data are expressed as mean ± standard deviation (SD). Statistical analyses were conducted using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparisons test to evaluate differences between treatment groups and controls. A p-value of <0.05 was considered indicative of statistical significance in all analyses.

3. Results and Discussion

3.1. Px NPs Optimization

To optimize Px NPs, formulations with varying drug inputs (5%–30%) were prepared, using B NPs as a control. Key parameters, particle size, PDI, %EE, and %DL, were evaluated to identify the most suitable formulation for brain-targeted delivery.

3.1.1. Particle Size and PDI

DLS analysis revealed that all nanoparticle formulations exhibited sizes below 100 nm, which is favorable for cellular uptake and brain biodistribution. ,,, As shown in Figure a, particle sizes ranged from 77.8 ± 1.0 nm for blank NPs to a maximum of 89.9 ± 1.5 nm at 15% drug input. Interestingly, formulations with higher drug input (20%, 25%, and 30%) showed slightly smaller particle sizes compared to those with lower inputs (5–15%). This inverse trend, while atypical for chitosan–TPP systems, may be attributed to increased ionic strength at higher Px concentrations. The additional counterions introduced by Px could screen the protonated amine groups of chitosan, reduce intramolecular electrostatic repulsion, and promote polymer chain compaction, ultimately resulting in smaller, more condensed nanoparticles. PDI values remained low across all formulations, ranging from 0.23 ± 0.01 to 0.28 ± 0.02, indicating uniform particle populations.

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Optimization of Px NPs. (n = 3). (a) Particle size and PDI for Px NPs with varying Px concentrations (5%, 10%, 20%, 30%) compared to the B NPs (0%). (b) %EE and %DL across varying Px inputs from 5% to 30%.

3.1.2. %EE and %DL

%EE and %DL were evaluated to assess Px incorporation into chitosan NPs. As shown in Figure b, %EE ranged from 25.3 ± 5.2% at 5% drug input to a peak of 30.8 ± 4.8% at 10%, but remained relatively constant with further increases in drug input up to 30%. This plateau, which deviates from typical chitosan–TPP systems, may be due to the hydrophilic and low molecular weight nature of Px·2HCl, limiting its affinity for the polymeric matrix and favoring its partition into the aqueous phase during NP formation. Additionally, strong chitosan–TPP ionic cross-linking may outcompete drug–polymer interactions at higher inputs, capping entrapment efficiency. Similar behavior has been reported in NP systems loaded with small, water-soluble drugs. − In contrast, %DL increased proportionally with drug input, from ∼3% at low levels to 8.3 ± 0.7% at 30%, indicating enhanced drug payload per unit mass without substantial gain in %EE. Among the formulations, the 25% input condition achieved a favorable profile, with %DL of 7.1 ± 0.7%, %EE of 28.2 ± 2.8%, and particle size of 82.2 ± 0.3 nm with low PDIparameters supportive of colloidal stability and efficient uptake. These characteristics suggest that the 25% formulation offers the most promising profile for further experiments.

3.2. Px NPs Characterization of the Optimized Formula

The 25% drug input Px NPs exhibited physicochemical properties indicative of suitability for drug delivery applications. Comprehensive characterization assessed particle size, surface charge, morphology, and release behavior. DLS analysis confirmed an average particle size of ∼85 nm with a low PDI, reflecting a narrow size distribution and uniform NP population (Figure a). The sub-100 nm size range is advantageous for promoting cellular uptake and favorable biodistribution. ,, Zeta potential was measured at 13.8 ± 1.3 mV, indicating a positively charged surface (Figure b). The cumulative release profile of Px from the optimized NPs was evaluated over 48 h in PBS (pH 7.4) at 37 °C to simulate physiological conditions. As shown in Figure c, a biphasic release pattern was observed, characterized by an initial burst release (∼50% within 6 h), followed by a sustained release phase extending to 48 h, reaching ∼85% cumulative release. The initial burst is attributed to the rapid diffusion of Px loosely associated with the NP surface, while the prolonged release phase reflects controlled diffusion of Px from the chitosan matrix. ,, This biphasic profile is desirable for maintaining therapeutic levels over extended periods, potentially enhancing bioavailability and reducing dosing frequency.

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Characterization of optimized Px NPs (25% Drug Input). (a) Size distribution is 85.2 ± 0.7 nm with a PDI of 0.26 ± 0.04. (b) Zeta potential measured using the same DLS system, showing an average of 13.8 ± 1.3 mV, which indicates particle stability. (c) Cumulative drug release profile of Px NPs over 48 h (n = 4). Approximately 85% cumulative release by the end of the observation period. (d) FE-SEM image of Px NPs at 30,000× magnification to observe morphology, showing round-shaped particles under 100 nm.

FE-SEM imaging further confirmed the morphological characteristics of Px NPs, revealing smooth, spherical particles with uniform size distribution consistent with DLS data (Figure d). The observed particle diameters remained below 100 nm, validating the controlled fabrication process.

3.3. Neuroprotective Effect of Px NPs on SH-SY5Y Model

The cytotoxicity of free Px, Px NPs, and B NPs was evaluated in SH-SY5Y cells prior to neuroprotection studies to ensure sample safety during the 1 h pretreatment period. As shown in Figure a, cell viability remained high across all tested concentrations of free Px and Px NPs, with only minor reductions observed. At 3.75 μg/mL, viability was 90.52 ± 0.07% for free Px and 87.58 ± 0.11% for Px NPs. At 7.5 μg/mL, Px NPs showed 96.31 ± 0.07% viability, while at 15 μg/mL, viability was 91.99 ± 0.02% for free Px and 93.73 ± 0.01% for Px NPs. These results indicate minimal cytotoxicity associated with Px, while B NPs showed no reduction, confirming the biocompatibility of the NP matrix. Importantly, all treatments maintained viability above the 70% threshold defined by ISO 10993-5, confirming that both free Px and Px NPs were noncytotoxic and suitable for subsequent neuroprotective evaluation. SH-SY5Y cells were utilized as an established in vitro PD model for many years, with oxidative stress induced by 1 mM H2O2 for 15 min to generate ROS-mediated injury. This treatment reduced cell viability to ∼50%, representing a significant decline compared to untreated controls (p < 0.05). Pretreatment with test samples for 1 h was conducted to assess neuroprotection via MTT assay. Statistical analysis was conducted using one-way ANOVA followed by Dunnett’s multiple comparisons test. As shown in Figure b, free Px exhibited no significant improvement in cell viability compared to the control group (p > 0.05), indicating a lack of neuroprotective effect under these conditions. In contrast, Px NPs demonstrated a concentration-dependent increase in viability, with 15 μg/mL showing a slight but statistically significant improvement compared to the untreated control group (p < 0.05), indicating a promising protective effect against ROS-induced damage in vitro. The limited efficacy of free Px observed in this study is consistent with previous reports, which highlight that Px activity depends not only on concentration but also on D2/D3 receptor expression levels. Given that SH-SY5Y cells express low levels of D2/D3 receptors, the cellular uptake of free Px may be insufficient to elicit a neuroprotective effect under these conditions. The superior performance of Px NPs may be attributed to multiple factors. The nanoscale size and positive surface charge of Px NPs likely promote enhanced cellular uptake via electrostatic interactions with negatively charged cell membranes. Additionally, chitosan’s intrinsic antioxidant properties, including H2O2 scavenging activity may synergistically reduce oxidative stress. , Together, these properties contribute to the observed neuroprotection.

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Cytotoxicity and neuroprotective effects of Px NPs, free Px, and B NPs on SH-SY5Y cells (PD model). (a) Cytotoxicity of samples against SH-SY5Y cells after 1 h of incubation. (b) Neuroprotective effect of Px NPs and free Px on H2O2-induced SH-SY5Y cells. (*p < 0.05).

3.4. Nanoparticles Biocompatibility

The biocompatibility of Px NPs was evaluated to ensure their potential for clinical translation and future therapeutic applications. Specifically, the hemolytic potential of Px NPs was assessed by evaluating their direct interaction with human RBCs, providing insight into their blood compatibility for potential systemic administration. The hemolysis assay was conducted by incubating RBCs with Px NPs for 3 h, followed by quantification of hemoglobin release. RBCs treated with 1% Triton X-100 served as the positive control, exhibiting 100% hemolysis, while RBCs treated with PBS served as the negative control, exhibiting 0% hemolysis. As shown in Figure a, slight hemolysis was observed at higher Px NPs concentrations, with 0.11 ± 0.03% at 0.5 mg/mL and 0.17 ± 0.07% at 1 mg/mL. Nevertheless, all hemolysis values remained well below the 2% threshold, confirming the nonhemolytic nature of all formulations. The excellent blood compatibility demonstrated by Px NPs supports their further development, including incorporation into microneedle platforms.

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Biocompatibility evaluation of nanoparticles. (a) The hemolysis activity of Px NPs was assessed by incubating RBCs with different concentration of Px NPs for 3 h at 37 °C. While (b) are the RBCs samples after tested which show no hemolyzed RBCs were observed among any samples except the positive control (+ve).

3.5. Microneedles Characterization

Px NPs MNs were successfully fabricated by concentrating Px NPs and dissolving 15% w/w CMC and 20% w/w PVA within the concentrated NPs at a 1:1 ratio. The morphology of the MNs is shown in Figure , displaying uniform conical-shaped microneedles with a total of 121 microneedles per 1 × 1 cm2 patch. SEM imaging (Figure ) revealed smooth surface morphology across all microneedle tips, with a needle height of 590–600 μm.

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An overview and close up images by SEM at 15×, 45× and 100× of B MNs (a) and Px NPs MNs (b).

The mechanical properties of the MNs were assessed to ensure that drug loading did not compromise mechanical strength. %Insertion efficiency and mechanical testing using a texture analyzer were performed to compare B MNs, Px NPs MNs, and free Px MNs. %Insertion efficiency was determined by counting methylene blue-stained sites on FTS skin following microneedle application (Figure b). Insertion efficiencies were 96.9% ± 0.2%, 96.1% ± 1.4%, and 95.6% ± 0.4% for B MNs, Px NPs MNs, and free Px MNs, respectively (Figure a). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test, which revealed no statistically significant differences among the groups (p > 0.05). Mechanical strength analysis revealed that all MN patches, upon the application of 32 N force via a texture analyzer, exhibited minimal tip deformation and no base fractures. The force–displacement profiles (Figure c) showed no sudden force drops, confirming mechanical integrity. Although slight variations in mechanical behavior were observed among the formulations, these did not significantly impact microneedle performance. High insertion efficiencies and the absence of mechanical failure support the robustness of Px NPs MNs for transdermal application. These findings suggest that the Px NPs MN formulation is promising for delivering therapeutic agents across the stratum corneum.

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Mechanical properties of different microneedle formulations. (a)­The percentage of insertion efficiency for B MNs, free Px MNs, and Px NPs MNs. (b) Representative images of microneedle insertion into FTS. The blue dots indicate points of penetration with a 0.5 cm scale bar. (c) Force–displacement curves and the microneedles patch after underwent the mechanical analysis (d) with a 0.2 cm scale bar.

3.6. Permeation Study

The permeation profiles of Px NPs MNs and free Px MNs were evaluated using FTS mounted on Franz diffusion cells to simulate transdermal drug delivery (Figure a). Both types of microneedles were applied to the skin surface and sealed with adhesive tape. As shown in Figure b, the cumulative permeation of Px through the skin was monitored over 72 h. Initially, both systems exhibited similar permeation patterns, with approximately 13 μg/cm2 of Px permeated within the first 12 h. Over time, the Px NPs MNs demonstrated superior sustained delivery, with a cumulative permeation of 43.3 ± 11.9 μg/cm2 after 72 h, compared to 33.1 ± 1.07 μg/cm2 for free Px MNs. This enhanced performance underscores the role of nanoparticles as drug reservoirs, facilitating prolonged and efficient transdermal delivery. These observations are consistent with prior ex vivo studies showing that nanoparticles can significantly enhance skin penetration compared to non-nanoparticle formulations.

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Permeation profile of Px NPs MNs and free Px MNs. (a) FTS was used as a skin model with Franz diffusion cell to study the permeation profile of the samples. (b) Comparison of the permeation efficacy between Px NPs MNs and Free Px MNs over 72 h under the same conditions (n = 3).

Moreover, compared to commercially available immediate-release Px tablets, which typically range from 0.125 to 1.5 mg per tablet, the proposed Px NPs MN system offers a promising alternative. Each microneedle patch exhibited an average drug loading of ∼0.12 mg/cm2. Based on this loading, a patch area ranging from approximately 1 to 11 cm2 could theoretically deliver systemically equivalent doses across the full clinical range. Importantly, the drug loading capacity of the MN system can be further optimized, either by increasing nanoparticle concentration or enhancing patch density, to accommodate higher dosing demands or extended wear time in future applications. The sustained-release characteristics of the Px NPs MN platform enable prolonged transdermal delivery from a single application, reducing dosing frequency compared to oral formulations. Additionally, this transdermal system circumvents first-pass metabolism and reduces the risk of swallowing difficulties, a critical advantage for patients with PD who commonly experience dysphagia. ,,, The dissolution kinetics of Px NP MNs, as shown in Figure S1, demonstrated moderately slow disintegration, with tip height reduced by approximately 85% within 120 min postinsertion, consistent with expected behavior for dissolvable MN systems. This gradual dissolution facilitates the timely release of embedded NPs into the skin. In parallel, the ex vivo permeation profile assessed using a Franz diffusion cell with full-thickness human skin showed that Px NP MNs achieved significantly higher cumulative Px permeation over 72 h compared to free Px MNs. This biphasic permeation pattern, characterized by an initial rapid release phase followed by sustained diffusion, reflects the combined contribution of MN dissolution and subsequent NP-mediated release from within skin layers. While the 48–72 h study duration is commonly employed in Franz cell-based evaluations of transdermal systems, we acknowledge that this model lacks physiological clearance mechanisms such as blood and lymphatic flow, which may result in prolonged apparent drug retention relative to in vivo conditions.

3.7. Microneedles Biocompatibility

Although CMC and PVA are recognized as biodegradable and biocompatible materials for biomedical applications, , their biosafety profiles must still be thoroughly evaluated to support future clinical development. In this study, the cytotoxicity and hemolytic potential of microneedle formulations were assessed to ensure their compatibility with biological systems. Cytotoxicity testing was performed on HDFn cells using eluted extracts from B MNs, Px NPs MNs, and B NPs MNs after 24 h incubation. As shown in Figure a, at 25% and 50% extract concentrations, cell viability for B MNs, B NPs MNs, and Px NPs MNs remained above 70%: 87.5 ± 10.6%, 103.6 ± 6.5%, 102.4 ± 7.9%, 89.9 ± 1.3%, 97.2 ± 3.1%, and 76.7 ± 2.3%, respectively. According to ISO 10993-5 (2009), a viability of 70% or higher is considered noncytotoxic, confirming the biocompatibility of all tested samples. Although minor reductions were noted, no significant cytotoxic effects were observed (p > 0.05), and the formulations were well tolerated by HDFn cells. ,

9.

9

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Biocompatibility evaluation of microneedles. (n = 3) (a) Cytotoxicity effects of B MNs, Px NPs MNs and B NPs MNs on HDFn cells at different concentrations were observed by MTT assay after 24 h of incubation. (b) The hemolytic activity of microneedles was evaluated by incubating RBCs with B MNs, Px NPs MNs, and B NPs MNs for 3 h at 37 °C.

The hemolysis assay further assessed blood compatibility by treating human RBCs with the microneedle extracts for 3 h. As shown in Figure b, all samples exhibited minimal hemolysis, remaining well below the 2% threshold for nonhemolytic classification. Together, the cytotoxicity and hemolysis assessments confirm that B MNs, Px NPs MNs, and B NPs MNs are biocompatible and safe for further therapeutic application. These findings support the feasibility of using these microneedles in transdermal drug delivery systems, offering a safe and effective platform for therapeutic agent administration.

4. Conclusion

The development of Px NPs MNs as a transdermal delivery platform for PD demonstrates strong potential. NPs prepared via ionic gelation exhibited an average size of 85.2 ± 0.7 nm with a PDI of 0.26 ± 0.04, and uniform spherical morphology confirmed by FE-SEM. These NPs enabled sustained drug release, with ∼85% of Px released over 48 h. Incorporation into dissolvable polymeric MNs preserved mechanical strength and achieved ∼100% insertion efficiency, as verified by force–displacement analysis. In vitro permeation studies showed that Px NPs MNs significantly enhanced transdermal delivery compared to free Px MNs. Biocompatibility and hemocompatibility evaluations confirmed the safety of the NPs and MN matrix in HDFn and SH-SY5Y cells, as well as RBCs. Additionally, Px NPs demonstrated superior neuroprotective effects at 15 μg/mL, which were not observed with free Px. In comparison to recent work by McGuckin et al. (2024), which utilized polymeric MNs with tablet-based reservoirs to achieve sustained plasma Px levels, our approach leverages chitosan-based NPs within dissolving MNs for improved drug loading, controlled release, and enhanced skin permeation. The system also benefits from the mucoadhesive and biocompatible properties of chitosan, supporting its scalability and translational relevance. While the present findings demonstrate favorable in vitro and ex vivo performance, in vivo studies remain necessary. Future work will investigate the pharmacokinetics, biodistribution, and therapeutic efficacy of Px NPs MNs in animal models of PD to validate their clinical potential and guide further optimization.

Supplementary Material

ao5c04299_si_001.pdf (88.4KB, pdf)

Acknowledgments

This research project is supported by Mahidol University (MU’s Strategic Research Fund: 2023) Grant Number MU-SRF-RS-02A/66.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c04299.

  • Dissolution kinetics of pramipexole-loaded microneedles in neonatal porcine skin (PDF)

The authors declare no competing financial interest.

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